Zonal heterogeneity in hepatic functions is well established both in vivo and in vitro, and is an important feature of the liver with implications fr zonal drug toxicity. However, how various factor gradients within the liver interact to modulate zonal hepatic functions in the human liver, and what role liver stromal cells play in such modulation are not well understood. Due to significant species-specific differences in liver functions, inducing and studying factors that regulate zonation in primary human liver cells is critical to elucidate similarities and differences with their animal counterparts for enabling a continuum of in vitro and in vivo investigations. However, in vitro models of human liver zonation are lacking, especially those that evaluate the long-term effects of factor gradients and stromal interactions on zonal functions in primary human hepatocytes. In order to address the aforementioned gap in knowledge, here we propose to investigate the effects of liver- relevant factor gradients and liver stromal cells on long-term functions of primary human hepatocytes in culture. We will utilize microfluidics to enable minimal use of limited primary human liver cells, and create independent, complex and overlapping gradients of soluble gradients onto multicellular liver cultures. We will assess the effects of gradients (O2, hormones) generated by our device on liver functions thought to be zonated in vivo, in the presence or absence of liver stromal cells. Results in vitro will be compared to mechanisms known in vivo. Ultimately, creating approaches and design rules for making a heterogeneous and stable population of human liver cells will allow recapitulation of this important liver feature in systems designed for drug screening, clinical use (i.e. bioartificial liver devices), and stem cell differentiation.